Pomegranate peel, chokeberry leaves and Ironwort extract as novel natural inhibitors of amylin aggregation and cellular toxicity in pancreatic ß cells.

Impeding or reducing human amylin aggregation and/or its toxicity can be key to preventing pancreatic islet amyloidosis and ß-cell loss in patients with Type 2 Diabetes Mellitus (T2DM). Here, Punica granatum (pomegranate) peel, Sideritis raeseri (ironwort) and Aronia melanocarpa (chokeberry) leaf extracts, were tested for their novel anti-aggregative and antitoxic properties in human amylin (hIAPP) treated rat pancreatic insulinoma (INS) cells. The protein aggregation (Th-T) assay revealed an inhibitory trend of all three plant extracts against amylin aggregates. In agreement with this finding, pomegranate peel and ironwort extracts effectively prevented the transition of hIAPP from disordered, random coil structures into aggregation prone ß-sheet enriched molecular assemblies, revealed by CD spectroscopy. Consistent with their anti-aggregative action, all three extracts prevented, to various degrees, reactive oxygen species (ROS) accumulation, mitochondrial stress, and, ultimately, apoptosis of INS cells. Collectively, the results from this study demonstrate effectiveness of natural products to halt hIAPP aggregation, redox stress, and toxicity, which could be exploited as novel therapeutics against amylin-derived islet amyloidosis and ß-cell stress in T2DM.

ß-Carboline Alkaloids Resist the Aggregation and Cytotoxicity of Human Islet Amyloid Polypeptide.

ß-Carboline alkaloids have a variety of pharmacological activities, such as antitumor, antibiosis and antidiabetes. Harmine and harmol are two structurally similar ß-carbolines that occur in many medicinal plants. In this work, we chose harmine and harmol to impede the amyloid fibril formation of human islet amyloid polypeptide (hIAPP) associated with type 2 diabetes mellitus (T2DM), by a series of physicochemical and biochemical methods. The results indicate that harmine and harmol effectively prevent peptide fibril formation and alleviate toxic oligomer species. In addition, both small molecules exhibit strong binding affinities with hIAPP mainly through hydrophobic and hydrogen bonding interactions, thus reducing the cytotoxicity induced by hIAPP. Their distinct binding pattern with hIAPP is closely linked to the molecular configuration of the two small molecules, affecting their ability to impede peptide aggregation. The study is of great significance for the application and development of ß-carboline alkaloids against T2DM.

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils.

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone but deposits as amyloid fibrils in more than 90% of patients with type II diabetes (T2D). Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14-37. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29 constitute the core of the hIAPP amyloid; (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G; (iii) explains why the six residue substitutions in rodent IAPP prevent aggregation; and (iv) suggests regions responsible for the observed hIAPP cross-seeding with ß-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors. Four of the designed peptides delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof of concept that the capping strategy can be used on full-length cryo-EM fibril structures.

Cichoric acid from witloof inhibit misfolding aggregation and fibrillation of hIAPP.

The misfolding, aggregation and fibrillation of human islet amyloid polypeptide (hIAPP) has been acknowledged as a hallmark event in type-II diabetes. Hence, inhibiting the misfolding, aggregation and fibrillation of hIAPP have been accepted as a vital factor to treat the disease. Here cichoric acid was extracted from witloof to explore its inhibition effects on misfolding, aggregation and fibrillation of hIAPP. Thioflavin-T (ThT) fluorescence assay, dynamic light scattering (DLS) and atomic force microscopy (AFM) images showed that cichoric acid inhibited the aggregation and fibrillation of hIAPP in a dosage-dependent manner. Circular dichroism (CD) spectra showed that cichoric acid inhibited the misfolding of hIAPP from unfolded to ß-sheet. Molecular docking and further experiments revealed interactions between hIAPP and cichoric acid. Cichoric acid could bind to K1 and R11 of hIAPP via electrostatic interaction. In addition, cichoric acid could form π-π stacking with hIAPP residues F15 and F23. These interactions inhibited the misfolding, aggregation and fibrillation of hIAPP. These results, together with cichoric acid's good cytocompatibility and significant protective effects in hIAPP lesioned cell models, not only showed that cichoric acid could be used to fight against amyloidosis, but also brought a new perspective for Chinese herbal medicine as natural compound's medical potential.

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic ß-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with ß-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic ß-cells. Furthermore, AZD supplementation in pancreatic ß-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

Mitigating Human IAPP Amyloidogenesis In Vivo with Chiral Silica Nanoribbons.

Amyloid fibrils generally display chirality, a feature which has rarely been exploited in the development of therapeutics against amyloid diseases. This study reports, for the first time, the use of mesoscopic chiral silica nanoribbons against the in vivo amyloidogenesis of human islet amyloid polypeptide (IAPP), the peptide whose aggregation is implicated in type 2 diabetes. The thioflavin T assay and transmission electron microscopy show accelerated IAPP fibrillization through elimination of the nucleation phase and shortening of the elongation phase by the nanostructures. Coarse-grained simulations offer complementary molecular insights into the acceleration of amyloid aggregation through their nonspecific binding and directional seeding with the nanostructures. This accelerated IAPP fibrillization translates to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental, and behavioral assays. This study has implicated the potential of employing chiral nanotechnologies against the mesoscopic enantioselectivity of amyloid proteins and their associated diseases.

Diabetes Drug Discovery: hIAPP1-37 Polymorphic Amyloid Structures as Novel Therapeutic Targets.

Human islet amyloid peptide (hIAPP1-37) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1-37) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1-37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1-37. Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A-F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.

Amylin Amyloid Inhibition by Flavonoid Baicalein: Key Roles of Its Vicinal Dihydroxyl Groups of the Catechol Moiety.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in ß-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic ß-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic ß-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic ß-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and ß-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent ß-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

Promotion or Inhibition of Islet Amyloid Polypeptide Aggregation by Zinc Coordination Depends on Its Relative Concentration.

Zinc is reported to play a complex role in islet amyloid polypeptide (IAPP) aggregation, which is associated with ß-cell death in type II diabetes (T2D). Depending on their relative concentrations in vitro, zinc could either promote or inhibit IAPP aggregation. Interestingly, genomewide association studies suggested both positive and negative correlations between T2D risks and activities of a ß-cell-specific zinc transporter upon mutations, which determines zinc concentration in vivo. To decipher the effect of zinc coordination on IAPP aggregation, we performed atomistic discrete molecular dynamics simulations to systemically study aggregation propensities of zinc-coordinated IAPP oligomers with different molecular weights (MWs), whose populations are determined by zinc concentration. We find that at low zinc:IAPP stoichiometry, zinc coordination promotes aggregation by forming high-MW oligomers. The aggregation is inhibited when the stoichiometry increases and zinc binds individual peptides. Our computationally derived predictions are validated by the complementary thioflavin-T fluorescence assay measuring the dependence of IAPP aggregation on a wide range of zinc concentrations. Our combined computational and experimental study offers detailed mechanistic insight into the complex role of zinc on IAPP aggregation and T2D development.

Proanthocyanidins are the major anti-diabetic components of cinnamon water extract.

Cinnamon consumption has been found to associate with the attenuation of diabetes mellitus. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as a causative factor of type 2 diabetes mellitus (T2DM). Here, we investigated whether cinnamon has any beneficial effect on the toxic aggregation of hIAPP. We found that cinnamon water extract (CWE) inhibited the amyloid formation of hIAPP in a dose-dependent manner, and identified proanthocyanidins as the major anti-amyloidogenic compounds of CWE. Proanthocyanidins affected the secondary structures of hIAPP and delayed the structural transition from unstructured coils to ß-sheet-rich structures. Further studies showed that proanthocyanidins not only inhibited the formation of hIAPP oligomers, but also significantly attenuated the membrane damaging and cytotoxic effects caused by the hIAPP aggregation. Together, these results suggest a possible way by which cinnamon shows beneficial effects on T2DM, and indicate a potential pharmacological usage of proanthocyanidins as an anti-diabetic drug candidate.

Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptide and protects pancreatic beta-cells against cytotoxicity.

The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of type 2 diabetes mellitus (T2DM). Salvia miltiorrhiza (Danshen), one of the most commonly used of traditional Chinese medicines, is often used in Compound Recipes for treating diabetes, however with unclear mechanisms. Since salvianolic acid B (SalB) is the most abundant bioactive ingredient of salvia miltiorrhiza water-extract. In this study, we tested whether SalB has any effect on the amyloidogenicity of hIAPP. Our results clearly suggest that SalB can significantly inhibit the formation of hIAPP amyloid and disaggregate hIAPP fibrils. Furthermore, photo-crosslinking based oligomerization studies suggest SalB significantly suppresses the toxic oligomerization of hIAPP monomers. Cytotoxicity protection effects on pancreatic INS-1 cells by SalB were also observed using MTT-based assays, potentially due to the inhibition on the membrane disruption effects and attenuated mitochondria impairment induced by hIAPP. These results provide evidence that SalB may further be studied on the possible pharmacological treatment for T2DM.

Alternative pathways of human islet amyloid polypeptide aggregation distinguished by (19)f nuclear magnetic resonance-detected kinetics of monomer consumption.

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real time. We show that alternative pathways of aggregation can be distinguished by using (19)F nuclear magnetic resonance (NMR) to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable buildup of nonfibrillar intermediates, in contrast to the well-studied Aß and α-synuclein proteins. To further develop the usage of (19)F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using (19)F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes nonfibrillar large aggregates during fibrillogenesis.

Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus.

Global epidemic studies have suggested that coffee consumption is reversely correlated with the incidence of type 2 diabetes mellitus (T2DM), a metabolic disease. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Coffee extracts have three major active components: caffeine, caffeic acid (CA), and chlorogenic acid (CGA). In this study, the effects of these major coffee components, as well as dihydrocaffeic acid (DHCA) (a major metabolite of CGA and CA), on the amyloidogenicity of hIAPP were investigated by thioflavin-T based fluorescence emission, transmission electronic microscopy, circular dichroism, light-induced cross-linking, dynamic light scattering, and MTT-based cell viability assays. The results suggest that all components show varied inhibitory effects on the formation of toxic hIAPP amyloids, in which CA shows the highest potency in delaying the conformational transition of the hIAPP molecule with the most prolonged lag time, whereas caffeine shows the lowest potency. At a 5-fold excess molar ratio of compound to hIAPP, all coffee-derived compounds affect the secondary structures of incubated hIAPP as suggested by the circular dichroism spectra and CDPro deconvolution analysis. Further photoinduced cross-linking based oligomerization and dynamic light scattering studies suggested CA and CGA significantly suppressed the formation of hIAPP oligomers, whereas caffeine showed no significant effect on oligomerization. Cell protection effects were also observed for all three compounds, with the protection efficiency being greatest for CA and least for CGA. These findings suggest that the beneficial effects of coffee consumption on T2DM may be partly due to the ability of the major coffee components and metabolites to inhibit the toxic aggregation of hIAPP.

Evaluation of aluminium, manganese, copper and selenium effects on human islets amyloid polypeptide hormone aggregation.

Islet amyloid formation causes destruction of insulin-producing beta-cells of the pancreas. The subsequent lack of insulin leads to increased blood and urine glucose. In this research, the fluorimetric assay was used to examine the effects of aluminium and some nutritionally essential trace elements including, manganese, copper and selenium on amyloid formation of human peptide of amylin under near-physiological circumstances. Results obtained from in vitro study showed that after 120 h incubation by shaker incubator in 37 degrees C, copper and selenium at 8 microM inhibited amylin 8 microM from amyloid fibril formation by 22.1 and 11.3%, respectively (p<0.05) while the similar values of either aluminium and manganese promoted the formation of beta-pleated sheet structure by 19.3 and 13.2% respectively (p<0.05). If islet amyloid is cytotoxic to beta-cells then copper and selenium may be able to protect these cells against degeneration in diabetic patients especially in type 2 diabetes mellitus.